By means of endogenous circadian (approx. 24 hr) "clocks" or pacemakers that can be synchronized to the daily and seasonal changes in external time cues, most notably visible light and ambient temperature, life forms anticipate environmental transitions, perform activities at biologically advantageous times during the day and undergo characteristic seasonal responses. Core features of many, if not all, circadian clocks are transcriptional feedback loops that generate daily cycles in the levels of clock mRNAs. However, posttranslational mechanisms make significant contributions to the temporal regulation of clock protein levels and activities. Time-of-day specific differences in phosphorylation that result in phase-specific changes in protein stability appear to be a common strategy in generating daily cycles in clock protein abundance. The main goal of this proposal is to better understand the role of clock protein phosphorylation in circadian function using Drosophila as an animal model system. In particular, a conserved feature of animal clocks is that PERIOD (PER) proteins undergo daily rhythms in phosphorylation that are essential for the normal progression of the time-keeping mechanism. Indeed, it is likely that the "ticking" of animal clocks is based on daily changes in the phosphorylated status of PER proteins. However, it is not clear what roles different phosphorylation sites play in regulating PER levels and activity. In this proposal, phosphorylation sites on the Drosophila PER protein will be identified and their specific contributions to the underlying clock mechanism determined. Understanding how phosphorylation regulates PER metabolism and activity has potential health implications as evidenced from the fact that mutations altering the phosphorylation of human PER2 or a key kinase that phosphorylates PER proteins (called CKI4) are linked to several familial sleep disorders. Moreover, recent findings in cyanobacteria suggest that the most basic and ancient building block of circadian clocks is a biochemical oscillator based on time-of-day specific phosphorylation of one or more key clock proteins. Thus, our studies should reveal novel insights on the biochemical basis for circadian clocks, which could lead to better treatments for disorders associated with clock dysfunction in humans. PUBLIC HEALTH RELEVANCE: Humans exhibit daily changes in physiology and behavior, such as our wake-sleep cycles, that are controlled by a network of specialized cells called circadian clocks. A key gear in the timing mechanism of these cellular clocks is a protein called PERIOD that when mutated can lead to severe sleep disorders and other health related problems. This proposal will investigate how PER proteins contribute to the "ticking" of the clock.